Procedure for the production of ethanol from lignocellulosic biomass using a new heat-tolerant yeast

Abstract

It includes the stages of grinding the lignocellulosic biomass to a size of 15-30 mm, subjecting the product obtained to steam explosion pre-treatment at a temperature of 190-230° C. for between 1 and 10 minutes in a reactor ( 2 ), collecting the pre-treated material in a cyclone ( 3 ) and separating the liquid and solid fractions by filtration in a filter press ( 9 ), introducing the solid fraction in a fermentation deposit ( 10 ), adding a cellulase at a concentration of 15 UFP per gram of cellulose and 12.6 International Units of β-glucosidase enzyme dissolved in citrate buffer pH 4.8, inoculating the fermentation deposit ( 10 ) with a culture of the heat-tolerant bacteria Kluyveromyces marxianus CECT 10875, obtained by chemical mutagenesis from strain DER-26 of Kluyveromyces marxianus and shaking the mixture for 72 hours at 42° C.

1 . Procedure for the production of ethanol from lignocellulosic biomass characterised in that it includes the stages of: 
 Grinding the lignocellulosic biomass Subjecting the ground lignocellulosic biomass to steam explosion pre-treatment, maintaining at a pressure of between 1 and 3 MPa and a temperature between 190 and 230° C., for a period of time of between 1 and 10 minutes, depending on the type of material used and later provoking rapid de-pressurisation.    Collecting the pre-treated material and separating the liquid and solid fractions by filtration, and introducing the solid fraction in the fermentation deposit ( 10 ).    Adding a cellulase to the fermentation deposit ( 10 ) in a concentration of 15 UFP per gram of cellulose and 12.6 International Units of β-glucosidase enzyme. Inoculating the fermentation deposit ( 10 ) with a suspension of a culture of the heat-tolerant yeast  Kluyveromyces marxianus  CECT 10875. Shaking the mixture for 72 h at 42° C. Determining the concentration of ethanol and residual sugars in the mix, once the reaction is complete.    
     
     
         2 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the particle size of the lignocellulosic biomass after grinding is between 15 and 30 mm.  
     
     
         3 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the culture of  Kluyveromyces marxianus  CECT 10875 is obtained by chemical mutagenesis from the DER-26 strain of  Kluyveromyces marxianus.    
     
     
         4 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the humidity content of the lignocellulosic biomass is between 10 and 15%.  
     
     
         5 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the solid fraction that is introduced into the fermentation deposit has a solid/liquid ratio that varies between 8 and 15% (w/v).  
     
     
         6 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the cellulase is CELLUCLAST 1.5L, from the NOVO-NORDISK company, and the β-glucosidase enzyme is NOVOZYME 188, from the NOVO-NORDISK company.  
     
     
         7 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the cellulase and the β-glucosidase are dissolved in citrate buffer pH 4.8.  
     
     
         8 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the  Kluyveromyces marxianus  CECT 10875 inoculant is at a concentration of 10 v/v.  
     
     
         9 . Procedure for the production of ethanol from lignocellulosic biomass, in accordance with  claim 1 , characterised in that the mixture is shaken at 150 r.p.m.